Skin acts as the first line of defense against invading microorganisms. It not only provides a physical barrier to pathogen entry, but also initiates vigorous innate immune responses upon sensing danger signals. Upon invasion, viruses release or produce viral nucleic acids, which are potent stimulators for the host skin immune system that trigger host antiviral innate immunity and inflammation. How the skin immune system detects viruses and restricts their spread is not well understood. Eczema herpeticum and eczema vaccinatum are two dangerous viral-associated skin diseases (caused by two DNA viruses, herpes virus and poxvirus, respectively) that occur in patients with atopic dermatitis, a condition associated with defects in skin barrier function and antiviral innate immunity. Our previously published results demonstrate that murine primary keratinocytes (KCs) can mount vigorous innate immune responses to cytosolic dsRNA. In addition, we found that the STING-mediated cytosolic DNA- sensing pathway and the MDA5-mediated cytosolic dsRNA-sensing pathway play critical and non-redundant roles in restricting viral replication in immortalized murine embryo fibroblasts (MEFs). These preliminary results form the basis of this proposal. We hypothesize that the type I IFN pathway plays an important role in modulating viral replication and therefore host range determination. Our aims are as follows: Aim 1a. Assess the innate immune responses of human and murine keratinocytes (KCs) and fibroblasts to cytosolic nucleic acid stimulation; Aim 1b. Delineate the type I IFN signature in murine skin keratinocytes and fibroblasts by using RNA-seq analysis in wild-type cells or cells deficient of the cytosolic nucleic acid-sensing pathways in response to immune agonists; and Aim 2. Determine the roles of the cytosolic nucleic acid-sensing pathways in host restriction of viral replication in murine and human adult fibroblasts. We will use murine primary KCs and fibroblasts from WT and knock-out mice to test our hypothesis that attenuated vaccinia virus-induced activation of the type I IFN pathway in murine KCs and fibroblasts requires the cytosolic dsRNA- and the DNA- sensing pathways. We will also compare viral induction of innate immunity in human KCs and fibroblasts with that in murine cells. We will build upon our experience using RNA-seq technology to elucidate type I IFN signature induced by stimulation of nucleic acids in skin KCs and fibroblasts. In addition, we will use state-of- art genome-editing technology CRISPR/Cas to generate single and double knock-out of genes in the cytosolic nucleic acid-sensing pathways in human primary skin fibroblasts, which will allow us to decipher their roles in antiviral immunity i human fibroblasts. Our proposed study will address the gaps in knowledge that are highly relevant to skin immunity, and our results will have significant impact on the development of skin-targeted therapy for viral-associated skin diseases.